Methods for making mixed allergen compositions

ABSTRACT

Methods of making mixed allergen products are provided, wherein the mixed allergen products are substantially free of replication viable organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/795,877, filed Jan. 23, 2019, and to U.S.Provisional Patent Application No. 62/865,473, filed Jun. 24, 2019, thedisclosure of which is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND

Allergy is a disorder of the immune system characterized by theoccurrence of allergic reactions to normally harmless environmentalsubstances. Allergies are caused by allergens, which may be present in awide variety of sources including, but not limited to, pollens or otherplant components, dust, molds or fungi, foods, additives, latex,transfusions, animal or bird danders, insect venoms, radiocontrastmedium, medications, or chemicals. Common allergic reactions includeeczema, hives, hay fever, asthma, and reactions to venoms. Mildallergies, like hay fever, are highly prevalent in the human populationand cause symptoms such as allergic conjunctivitis, itchiness, and runnynose. In some people, severe allergies to environmental allergens,dietary allergens, or medication may result in life-threateninganaphylactic reactions if left untreated.

A food allergy is an adverse immune response to a food allergen, forexample, a food protein. Common food allergens are found in shellfish,peanuts, tree nuts, fish, milk, eggs, soy and fresh fruits such asstrawberries, mangoes, bananas, and apples. Immunoglobulin E(IgE)-mediated food allergies are classified as type-I immediatehypersensitivity reactions. These allergic reactions have an acute onset(from seconds to one hour) and the accompanying symptoms may include:angioedema (soft tissue swelling of the eyelids, face, lips, tongue,larynx and trachea); hives; itching of the mouth, throat, eyes, andskin; gastrointestinal symptoms such as nausea, vomiting, diarrhea,stomach cramps, and abdominal pain; rhinorrhea or nasal congestion;wheezing; shortness of breath; difficulty swallowing; and anaphylaxis, asevere, whole-body allergic reaction that can result in death. It isestimated that 1 out of 12 children under 21 years of age have adiagnosed food allergy, and over $24 billion is spent per year on healthcare costs for food allergic reactions, largely due to about 90,000emergency room visits per year in the U.S. alone due to food-inducedanaphylaxis. Moreover, deaths occur every year due to fatal foodallergies.

Accordingly, there exists a need in the art for allergen compositionsthat can prevent and/or treat allergies, and methods for making allergencompositions to prevent and/or treat allergies.

SUMMARY

This disclosure is directed, at least in part, to a method of making amixed allergen product substantially free of replication viableorganisms. For example, in certain embodiments, the method comprises:providing bulk mixed allergen material comprising 2 to 20 individualcomplete food allergens; and sterilizing the bulk mixed allergenmaterial by dry heat sterilization, thereby obtaining the mixed allergenproduct.

In certain embodiments, a method further comprises providing 2 to 20individual complete food allergens and blending the 2 to 20 individualcomplete food allergens together to obtain the bulk mixed allergenmaterial.

In other embodiments, the disclosure provides a method, wherein the 2 to20 individual complete food allergens are selected from the groupconsisting of almond, cashew, cod, egg, hazelnut, milk, oat, peanut,pecan, pistachio, salmon, sesame, shrimp, soy, walnut, and wheat.

In another embodiment, the disclosure provides a method, wherein theblending further comprises blending the 2 to 20 individual complete foodallergens with one or more than one bulking agent.

In certain embodiments of the present disclosure, blending comprisesloading 2 to 20 individual complete food allergens into a ribbon blenderset at about 10 Hz to about 50 Hz for about 10 minutes to about 50minutes. In other embodiments, the bulk mixed allergen material isdischarged into lined drums after blending.

In certain embodiments, a method of the present disclosure furthercomprises filtering the mixed allergen product. In some embodiments,filtering comprises passing the mixed allergen product through a screenranging from a #5 mesh (US) screen to a #10 mesh (US) screen on a shakerfor about 1 minute to about 10 minutes.

In some embodiments, a method of the present disclosure furthercomprises passing the mixed allergen product through a rare earthmagnet, metal detector, and metal separator.

In certain embodiments, a described method of the present disclosureprovides applying dry heat sterilization, wherein the dry heatsterilization is at a temperature of about 65° C. to about 175° C. forabout 18 hours to about 72 hours. In some embodiments, the dry heatsterilization comprises using a sterilization system having a furnaceand a heating chamber, wherein the furnace provides about 250,000 BTUsto about 750,000 BTUs of heat to the heating chamber. In certainembodiments, the bulk mixed allergen material is first packaged intothermoresistant packaging before dry heat sterilization. In certainembodiments, temperature probes are placed with the bulk mixed allergenmaterial during sterilizing to monitor the temperature of the dry heatsterilization.

In certain embodiments of the present disclosure, the mixed allergenproduct has at least about a 1 log reduction to at least about a 4 logreduction in aerobic bacterial organism plate counts as compared tocorresponding bulk mixed allergen material that has not been sterilized.In some embodiments, the mixed allergen product has less than about10,000 CFU/g of aerobic bacterial organisms. In other embodiments, themixed allergen product has less than about 10 CFU/g of coliforms,Escherichia coli, Enterobacteriaceae, mold, Staphylococcus aureus, andyeast. In certain embodiments, the mixed allergen product has less thanabout 10 CFU/g of Salmonella. In certain embodiments, the mixed allergenproduct has less than about 10 CFU/g of Cronobacter. In furtherembodiments, the mixed allergen product is negative in PCR-based assaysfor Listeria species, Cronobacter species and Salmonella species per 25g of mixed allergen product, 25 g of mixed allergen product, and 375 gof mixed allergen product, respectively.

In some embodiments of the present disclosure, the mixed allergenproduct has a fat content of about 10% to about 20% by weight.

In other embodiments, protein integrity of the mixed allergen product issubstantially similar to protein integrity of corresponding bulk mixedallergen material that has not been sterilized. In some embodiments, theprotein integrity of the mixed allergen product is determined bySDS-PAGE.

In certain embodiments, the mixed allergen product has a protein contentof about 40% to about 50% by weight.

In certain embodiments, the mixed allergen product has about 1% to about10% moisture by weight. In other embodiments, the mixed allergen producthas a water activity of about 0.1 to about 0.6.

In certain embodiments of the present disclosure, the mixed allergenproduct comprises less than about 10% by weight of particles having adiameter greater than 500 m.

In some embodiments, the mixed allergen product is darker in color ascompared to corresponding bulk mixed allergen material that has not beensterilized. In other embodiments, the mixed allergen product has asubstantially similar taste profile as compared to corresponding bulkmixed allergen material that has not been sterilized.

In certain embodiments, a described method of the present disclosurecomprises one or more than one further application of dry heatsterilization.

In an alternative embodiment, the present disclosure describes a methodof making a mixed allergen product substantially free of replicationviable organisms, comprising: providing 2 to 20 individual complete foodallergens; sterilizing each of the 2 to 20 individual complete foodallergens by dry heat sterilization, thereby obtaining 2 to 20sterilized individual complete food allergens; and blending the 2 to 20sterilized individual complete food allergens together, therebyobtaining the mixed allergen product.

In another embodiment, the present disclosure describes a method ofmaking a mixed allergen product substantially free of replication viableorganisms, wherein the method further comprises blending the mixedallergen product with one or more than one probiotic selected from thegroup consisting of Lactobacillus rhamnosus, Anaerostipes caccae, andBifidobactierium longum.

In yet another embodiment, the mixed allergen product is further blendedwith one or more than one prebiotic, for example, wherein the prebioticis fructooligosaccharide.

Also disclosed is a mixed allergen product substantially free ofreplication viable organisms prepared by any one of the methodsdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a macroscopic image of non-sterilized bulk mixed allergenmaterial sample and bulk mixed allergen material samples which have beenheated in a laboratory oven for 5 minutes at 95° C., 105° C., 110° C.,or 120° C.

FIG. 2A is a schematic showing the placement of pallets and sample boxescontaining thermoresistant bags of bulk mixed allergen material in a dryheat sterilization chamber for validation trial 1. B3 of pallets 3, 4and 12 have chamber probes placed inside the boxes for temperaturemonitoring. B4 of pallets 7 and 8 have chamber probes placed outside theboxes, and B4 of pallet 11 has a chamber probe placed inside the box.FIG. 2B is a schematic showing the placement of pallets and sample boxescontaining thermoresistant bags of bulk mixed allergen material in a dryheat sterilization chamber for validation trial 2. B3 of pallets 5 and14 have chamber probes placed inside the boxes for temperaturemonitoring. B4 of pallets 4 and 8 have chamber probes placed inside theboxes, and B4 of pallets 6 and 9 have chamber probes placed outside theboxes. Shaded boxes indicate empty boxes without thermoresistant bagscontaining bulk mixed allergen material. Asterisks (“*”) indicate boxeshaving two thermoresistant bags containing bulk mixed allergen material.“P” indicates boxes having a MadgeTech probe.

FIG. 3A is line graph showing the temperature profile of the dry heatsterilization chamber during validation trial 1. FIG. 3B is line graphshowing the temperature profile of the dry heat sterilization chamberduring validation trial 2. Each line represents the temperature readingsof a single chamber probe or MadgeTech probe.

DETAILED DESCRIPTION

Disclosed herein are methods of making a mixed allergen productsubstantially free of replication viable organisms.

By “replication viable organisms”, it is meant organisms that arecapable of multiplying, reproducing, propagating, and/or producingcolony forming units (CFU) on a plate culture.

By “sterilization”, it is meant a process that is capable ofsubstantially reducing the number of replication viable organisms.

As used herein, an “individual complete food allergen” refers to a foodsubstance containing all possible antigenic components for said foodsubstance (for example, allergenic proteins). Individual complete foodallergens may include, but are not limited to, unprocessed or processedfood substances, concentrated food substances, and isolated foodsubstances.

“Allergenic proteins”, as used herein, are antigenic components of foodallergens that are, either directly or indirectly, responsible foreliciting a biological allergic response. Allergenic proteins mayinclude, but are not limited to, nut proteins such as hazelnut proteins(e.g., Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11,Cor a 12, Cor a 13, and Cor a 14), cashew proteins (e.g., Ana o 1, Ana o2, and Ana o 3), pistachio proteins (e.g., Pis v 1, Pis v 2, Pis v 3,Pis v 4, and Pis v 5), walnut proteins (e.g., Jug r 1, Jug r 2, Jug r 3,Jug r 4, Jug r 5, Jug r 6, Jug r 7, and Jug r 8, Jug n1, Jug n 2, andJug n 4), pecan proteins (e.g., Car i 1, Car i 2, and Car i 4), almondproteins (e.g., Pru du 3, Pru du 4, Pru du 5, Pru du 6, and Pru du 8),peanut proteins (e.g., Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Arah 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13,Ara h 14, Ara h 15, Ara h 16, and Ara h 17), and brazil nut proteins(e.g., Ber e 1 and Ber e 2). Allergenic proteins may also include, butare not limited to, animal proteins such as egg proteins (e.g., Gal d 1,Gal d 2, Gal d 3, Gal d 4, Gal d 5, Gal d 6, Gal d 7, Gal d 8, Gal d 9,Gal d 10), milk proteins (e.g., Bos d 2, Bos d 3, Bos d 4, Bos d 5, Bosd, 6, Bos d 7, Bos d 8, Bos d 9, Bos d 10, Bos d 11, and Bos d 12),salmon proteins (e.g., Onc k 5, Sal s 1, Sal s 2, and Sal s 3), codproteins (e.g., pGad c 1, Gad m 1, Gad m 2, and Gad m 3), shrimpproteins (e.g., Cra c 1, Cra c 2, Cra c 4, Cra c 5, Cra c 6, Cra c 8,Lit v 1, Lit v 2, Lig v 3, Lit v 4, Met e 1, Pan b 1, Pen a 1, Pen i 1,Pen m 1, Pen m 2, Pen m 3, Pen m 4, and Pen m 6), and crab proteins(e.g., Cha f 1, Por p1, Scy p 2, Scy p 4, and Scy p 8). Allergenicproteins may further include, but are not limited to, non-nut plantproteins such as wheat proteins (e.g., Tri a 12, Tri a 14, Tri a 15, Tria 17, Tri a 18, Tri a 19, Tri a 20, Tri a 21, Tri a 25, Tri a 26, Tri a27, Tri a 28, Tri a 29, Tri a 30, Tri a 31, Tri a 32, Tri a 33, Tri a34, Tri a 35, Tri a 36, Tri a 37, Tri a 39, Tri a 40, Tri a 41, Tri a42, Tri a 43, Tri a 44, and Tri a 45), soy proteins (e.g., Gly m 1, Glym 1.0101, Gly m 2, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m 7, and Glym 8), chickpea proteins, sesame seed proteins (e.g., Ses i 1, Ses i 2,Ses i 3, Ses i 4, Ses i 5, Ses i 6, and Ses i 7), kiwi proteins (e.g.,Act c 1, Act c 5, Act c 8, Act c 10, Act d 1, Act d 2, Act d 3, Act d 4,Act d 5, Act d 6, Act d 7, Act d 8, Act d 9, Act d 10, Act d 11, Act d12, and Act d 13), carrot proteins (e.g., Dau c 1, Dau c4, and Dau c5),celery proteins (e.g., Api q 1, Api q 2, Api q 3, Api q 4, Api q 5, andApi q 6), stone fruit proteins (e.g., Pru ar 1, Pru ar 3, Pru av 1, Pruav 2, Pru av 3, Pru av 4, Pru p 1, Pru p 2, Pru p 3, Pru p4, Pru p 7,and Pru d 3), and oat proteins.

Presently disclosed, for example, is a method of making a mixed allergenproduct substantially free of replication viable organisms, comprising:providing bulk mixed allergen material comprising 2 to 20 individualcomplete food allergens; and sterilizing the bulk mixed allergenmaterial by dry heat sterilization, thereby obtaining the mixed allergenproduct.

In certain embodiments, bulk mixed allergen material comprises 2 to 20individual complete food allergens, for example, 4 to 20, 6 to 20, 8 to20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 2 to 18, 4 to 18,6 to 18, 8 to 18, 10 to 18, 12 to 18, 14 to 18, 16 to 18, 2 to 16, 4 to16, 6 to 16, 8 to 16, 10 to 16, 12 to 16, or 14 to 16 individualcomplete food allergens. For example, bulk mixed allergen material maycomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 individual complete food allergens. In a particular embodiment,bulk mixed allergen material comprises 15 or 16 individual complete foodallergens.

For example, in certain embodiments, the individual complete foodallergens are selected from the group consisting of nut, seed, legume,egg, dairy, fish and crustacean. In particular embodiments, theindividual complete food allergens are selected from the groupconsisting of almond, cashew, hazelnut, peanut, pecan, pistachio,walnut, sesame, soy, egg, milk, oat, wheat, cod, salmon, and shrimp. Incertain embodiments, the individual complete food allergens are almond,cashew, hazelnut, peanut, pecan, pistachio, walnut, sesame, soy, egg,milk, oat, wheat, cod, salmon, and shrimp. It is contemplated that theindividual complete food allergens may be provided as a meal, flour,powder, and/or protein concentrate.

In certain embodiments, a described method further comprises providing 2to 20 individual complete food allergens, and blending the 2 to 20individual complete food allergens together to obtain bulk mixedallergen material. It will be appreciated that two or more individualcomplete food allergens may be provided in combination prior toblending. For example, 2 to 20, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12to 20, 14 to 20, 16 to 20, 18 to 20, 2 to 18, 4 to 18, 6 to 18, 8 to 18,10 to 18, 12 to 18, 14 to 18, 16 to 18, 2 to 16, 4 to 16, 6 to 16, 8 to16, 10 to 16, 12 to 16, or 14 to 16 individual complete food allergensmay be provided in combination prior to blending. In a further example,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20individual complete food allergens may be provided in combination priorto blending.

It will be appreciated that blending may comprise one or more than oneblending steps to obtain bulk mixed allergen material. For example,individual complete food allergens may be blended in 1 to 19, 1 to 17, 1to 15, 1 to 13, 1 to 11, 1 to 9, 1 to 7, 1 to 5, or 1 to 3 blendingsteps. In a particular embodiment, blending comprises mixing individualcomplete food allergens in a single blending step.

As contemplated in the present disclosure, bulk mixed allergen materialis sterilized by dry heat sterilization. It will be appreciated thatsterilizing may further comprise one or more than one additionalapplication of dry heat sterilization, to inactivate heat-resistantspores.

In certain embodiments, dry heat may be supplied to a treatment chamberof a sterilization system within which bulk mixed allergen material isheld or is conveyed, wherein the treatment chamber may be heated to atemperature of about 65° C. to about 175° C. For example, in certainembodiments, the treatment chamber may be heated to about 65° C. toabout 80° C., about 65° C. to about 120° C., about 100° C. to about 150°C., or about 125° C. to about 175° C. In particular embodiments, thetreatment chamber may be heated to a temperature of about 65° C., about66° C., about 67° C., about 68° C., about 69° C., about 70° C., about71° C., about 72° C., about 73° C., about 74° C., about 75° C., about85° C., about 95° C., about 105° C., about 115° C., about 125° C., about135° C., about 145° C., about 155° C., about 165° C., or about 175° C.In further embodiments, dry heat may be applied to bulk mixed allergenmaterial for about 3 minutes to about 72 hours. In certain embodiments,for example, dry heat may be applied for about 3 minutes to about 72hours, about 30 minutes to about 72 hours, about 12 hours to about 72hours, about 18 hours to about 72 hours, about 24 hours to about 72hours, about 30 hours to about 72 hours, about 36 hours to about 72hours, about 42 hours to about 72 hours, about 48 hours to about 72hours, about 54 hours to about 72 hours, about 60 hours to about 72hours, or about 66 hours to about 72 hours. For example, in particularexamples, dry heat may be applied to bulk mixed allergen material forabout 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes,about 90 minutes, about 105 minutes, about 120 minutes, about 12 hours,about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours,or about 72 hours.

In certain embodiments, for example, dry heat may be provided to atreatment chamber of a sterilization system by multiple natural gasfired furnaces that provide about 250,000 BTUs to about 750,000 BTUs ofheat to the treatment chamber. For example, about 250,000 BTUs, about300,000 BTUs, about 350,000 BTUs, about 400,000 BTUs, about 450,000BTUs, about 500,000 BTUs, about 550,000 BTUs, about 600,000 BTUs, about650,000 BTUs, about 700,000 BTUs, or about 750,000 BTUs of heat may beprovided to the treatment chamber.

In a further embodiment, dry heat sterilization may be applied to bulkmixed allergen material packaged in thermoresistant packaging. As usedherein, “thermoresistant” is understood to mean that applying the sametemperature and duration of heat to the packaging material asconcurrently applied to the bulk mixed allergen material packagedtherein, does not cause changes in the packaging material that affectits integrity and functionality as a barrier to chemical or microbialcontamination. Furthermore, “thermoresistant” is understood to mean thatapplying the same temperature and duration of heat to the packagingmaterial as concurrently applied to the bulk mixed allergen materialpackaged therein, does not alter the packaging to cause a chemical inthe packaging to be added to the bulk mixed allergen material packagedtherein. For example, the thermoresistant packaging may include, but isnot limited to, bulk bags, pouches, or stickpacks. In a further example,the thermoresistant packaging may be placed in boxes and/or on palletsor racks within a heating chamber for dry heat sterilization.

As contemplated in a described method of the present disclosure,blending may comprise loading 2 to 20 individual complete food allergens(e.g., 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to20, 18 to 20, 2 to 18, 4 to 18, 6 to 18, 8 to 18, 10 to 18, 12 to 18, 14to 18, 16 to 18, 2 to 16, 4 to 16, 6 to 16, 8 to 16, 10 to 16, 12 to 16,or 14 to 16 individual complete food allergens) into a mechanicalblender or mixer. For example, the mechanical blender or mixer may beselected from, but not limited to: a ribbon blender; a paddle blender; aplow blender; a fluidizing blender; an intensive mixer; a vacuum mixer;a conical mixer; a rotary batch mixer; and a double shaft mixer. In aparticular embodiment, the individual complete food allergens are loadedinto a ribbon blender. Without wishing to be bound by theory, it isbelieved that blending by loading the individual complete food allergensinto a mechanical blender or mixer reduces grittiness, reduces thenumber of large particles, and increases homogeneity.

It will be appreciated that each individual complete food allergen maybe loaded into the mechanical blender or mixer in various amountsincluding, for example, but not limited to: about 5 kg to about 100 kg;about 10 kg to about 90 kg; about 15 kg to about 80 kg; about 20 kg toabout 70 kg; about 25 kg to about 60 kg; and about 30 kg to about 50 kg.It will be further appreciated that the mechanical blender or mixer mayaccommodate various total combined amounts of complete food allergens.In certain embodiments, for example, the mechanical blender or mixer mayaccommodate a total combined amount of complete food allergens of about100 kg to about 2200 kg or about 500 kg to about 2000 kg. For example,in some embodiments the mechanical blender or mixer may accommodate anamount of about 500 kg, about 600 kg, about 700 kg, about 800 kg, about900 kg, about 1000 kg, about 1100 kg, about 1200 kg, about 1500 kg,about 1600 kg, about 1700 kg, about 1800 kg, about 1900 kg, or about2000 kg of total combined complete food allergens.

It will also be appreciated that the settings of the mechanical blenderor mixer may be adjusted so as to achieve optimal blending and/or mixingof the individual complete food allergens. For example, the mechanicalblender may be set to about 10 Hz to about 50 Hz (e.g., about 15 Hz toabout 45 Hz, about 20 Hz to about 40 Hz, or about 25 Hz to about 35 Hz).The mechanical blender may also be set to operate for various lengths oftime. For example, the mechanical blender may blend the individual foodallergens for about 5 minutes to about 30 minutes, about 7.5 minutes toabout 25 minutes, or about 10 minutes to about 20 minutes. In aparticular embodiment, the mechanical blender or mixer is a 1000 kgcapacity ribbon blender and blends the individual complete foodallergens at 30 Hz for 15 minutes.

In certain embodiments, a method of the present disclosure furthercomprises blending the 2 to 20 individual complete food allergens withone or more than one bulking agents. Contemplated bulking agents mayinclude any bulking agent described herein. In certain embodiments, thebulking agent comprises a sugar, for example, sucrose, maltodextrin,trehalose, trehalose dihydrate, isomalt, mannitol, lactose, dextrose,fructose, raffinose, or any combination thereof. In certain embodiments,the bulking agent comprises maltodextrin, or sucrose, or a combinationthereof.

In certain embodiments, bulk mixed allergen material may be dischargedfrom a mechanical blender or mixer into lined drums before being loadedfor sterilization. In further embodiments, the bulk mixed allergenmaterial discharged into lined drums may be packaged in bags or pouches.

Also contemplated is a method of making a mixed allergen product,further comprising filtering a mixed allergen product to removeagglomerate particles. For example, a mixed allergen product may befiltered through a screen ranging from a #5 mesh (US) screen to a #10mesh (US) screen on a shaker for about 1 minute to about 10 minutes. Forexample, the screen may be a #5 mesh (US) screen, a #6 mesh (US) screen,a #7 mesh (US) screen, a #8 mesh (US) screen, a #9 mesh (US) screen, ora #10 mesh (US) screen. In a particular embodiment, the mixed allergenproduct is filtered through a screen ranging from a #5 mesh (US) screento a #10 mesh (US) screen on a shaker for about 3 minutes.

In certain embodiments, a method of making a mixed allergen product mayfurther comprise magnetically sifting a mixed allergen product to removemetallic particles. For example, magnetic sifting may comprise passingmixed allergen product through a rare earth magnet, a metal detector,and/or a metal separator. In a further example, the metal detector maydetect stainless steel particles at least about 2 mm to about 2.5 mm insize, ferrous particles at least about 1.5 mm to about 2 mm in size, andnon-ferrous particles with a sensitivity of about 1.5 to 2 parts permillion.

In further embodiments, a method of making a mixed allergen product maybe a continuous process, wherein the total completion time for themethod is about 3 minutes to about 72 hours, about 30 minutes to about72 hours, about 12 hours to about 72 hours, about 18 hours to about 72hours, about 24 hours to about 72 hours, about 30 hours to about 72hours, about 36 hours to about 72 hours, about 42 hours to about 72hours, about 48 hours to about 72 hours, about 54 hours to about 72hours, about 60 hours to about 72 hours, or about 66 hours to about 72hours. For example, in particular examples, the total completion timefor the method is about 30 minutes, about 45 minutes, about 60 minutes,about 75 minutes, about 90 minutes, about 105 minutes, about 120minutes, about 12 hours, about 18 hours, about 24 hours, about 30 hours,about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60hours, about 66 hours, or about 72 hours.

In methods of the present disclosure, sterilizing bulk mixed allergenmaterial by dry heat sterilization, produces a mixed allergen productthat is substantially free of replication viable organisms.

In certain embodiments, a mixed allergen product of the presentdisclosure is substantially free of replication viable bacteria, yeast,and/or molds. In some embodiments, a mixed allergen product may have: atleast about a 1 log reduction to at least about a 9 log reduction; atleast about a 1 log reduction to about a 4 log reduction; at least abouta 1 log reduction to about a 5 log reduction; at least about a 1 logreduction to about a 6 log reduction; at least about a 1 log reductionto about a 7 log reduction; at least about a 1 log reduction to about a8 log reduction, or at least about a 3 log reduction to about a 9 logreduction in aerobic bacterial organism plate counts as compared to acorresponding bulk mixed allergen material that has not been sterilized.For example, a mixed allergen product may have at least about a 1 logreduction, at least about a 2 log reduction, at least about a 3 logreduction, at least about a 4 log reduction, at least about a 5 logreduction, at least about a 6 log reduction, at least about a 7 logreduction, at least about a 8 log reduction, or at least about a 9 logreduction. in aerobic bacterial organism plate counts as compared to acorresponding bulk mixed allergen material that has not been sterilized.In a further example, a mixed allergen product has less than about100,000 CFU/g, 10,000 CFU/g, less than about 1,000 CFU/g, less thanabout 100 CFU/g, or less than about 10 CFU/g of aerobic bacterialorganisms. In another example, a mixed allergen product has less thanabout 100 CFU/g, less than about 10 CFU/g, or less than about 1 CFU/g ofcoliforms. In yet another example, a mixed allergen product may haveless than about 10 CFU/g of a pathogenic microbial strain. For example,a mixed allergen product has less than about 100 CFU/g, less than about10 CFU/g, or less than about 1 CFU/g of Escherichia coli. In a furtherexample, a mixed allergen product has less than about 100 CFU/g, lessthan about 10 CFU/g, or less than about 1 CFU/g of Enterobacteriaceae.In another example, a mixed allergen product has less than about 100CFU/g, or less than about 10 CFU/g of Salmonella. In another example, amixed allergen product has less than about 100 CFU/g, or less than about10 CFU/g of Cronobacter. In yet another example, a mixed allergenproduct is negative for Listeria species or Cronobacter species per 25 gsample of said mixed allergen product as determined by a PCR-basedassay. In another example, a mixed allergen product has less than about100 CFU/g, less than about 10 CFU/g, or less than about 1 CFU/g of mold.In yet another example, a mixed allergen product is negative forSalmonella species per 375 g sample of said mixed allergen product asdetermined by a PCR-based assay. In a further example, a mixed allergenproduct has less than about 100 CFU/g, less than about 10 CFU/g, or lessthan about 1 CFU/g of Staphylococcus aureus. In another example, a mixedallergen product has less than about 100 CFU/g, less than about 10CFU/g, or less than about 1 CFU/g of yeast.

In certain embodiments, a mixed allergen product of the presentdisclosure has a fat content of at least about 5% by weight. Forexample, a mixed allergen product may have a fat content of about 10% toabout 20% by weight. In some embodiments, for example, a mixed allergenproduct may have a fat content of about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,or about 20% by weight.

It is contemplated that applying dry heat sterilization to bulk mixedallergen material will not substantially alter protein integrity. Forexample, in certain embodiments, a sterilized mixed allergen product hassubstantially the same protein integrity as a corresponding bulk mixedallergen material that has not been sterilized. In a further example,protein integrity is determined by SDS-PAGE, wherein mixed allergenproduct has substantially similar protein bands, protein banddistribution, and protein band intensity as non-sterilized bulk mixedallergen material.

It is further contemplated that applying dry heat sterilization to bulkmixed allergen material will not substantially affect the ability ofbulk mixed allergen material to elicit an allergen effect upon beingconsumed by an individual. As used herein, “allergen effect” isunderstood to mean an immune reaction to one or more than one antigeniccomponent characterized by, but not limited to, immune cell activation,production of cytokines, and production of IgE. In certain embodiments,mixed allergen product has a substantially similar allergen effect uponbeing consumed by an individual as consumption of a substantiallysimilar protein amount of corresponding bulk mixed allergen materialthat has not been sterilized. In certain embodiments, an allergen effectis measured by the production of IgE or cytokines, or measuring immunecell activation in response to consumption of bulk mixed allergenmaterial or mixed allergen product by an individual. In otherembodiments, the allergen effect is measured by an in vitro immuneresponse, for example, measuring the production of IgE or cytokines, ormeasuring activation of immune cell cultures following treatment withbulk mixed allergen material or mixed allergen product.

In certain embodiments, a mixed allergen product of the presentdisclosure has a protein content of about 40% to about 50% by weight.For example, a mixed allergen product may have a protein content ofabout 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, or about 50% by weight.

In certain embodiments, a mixed allergen product of the presentdisclosure has about 1% to about 10% moisture by weight. For example, amixed allergen product may have about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% moistureby weight.

In certain embodiments, a mixed allergen product of the presentdisclosure has about 0.1 to about 0.6 water activity. “Water activity”is understood as the ratio between the vapor pressure of the mixedallergen product and the vapor pressure of distilled water underidentical conditions. It will be appreciated that water activity is ameasure of the water that is not bound to the molecules of the mixedallergen product and thus capable of supporting growth of bacteria,yeast and mold. Furthermore, it will be appreciated that water activitymay be measured using suitable electronic instruments such as moisturemeters, moisture-humidity meters, hygrometers, and relative humiditysystems.

In certain embodiments, a mixed allergen product of the presentdisclosure has less than about 10% by weight of particles having adiameter greater than 500 μm.

In yet another embodiment, a mixed allergen product of the presentdisclosure has a substantially similar taste profile as compared tocorresponding bulk mixed allergen material that has not been sterilized.For example, a mixed allergen product may have one or more than oneflavor selected from, but not limited to, savory, nutty, marine,roasted, and brown. In a further example, a mixed allergen product doesnot have any off-flavors from processing.

In another embodiment, a method of making a mixed allergen productsubstantially free of replication viable organisms is contemplated,wherein 2 to 20 individual food allergens are sterilized by dry heatsterilization before blending to obtain a mixed allergen product.

In certain embodiments, a method of making a mixed allergen productfurther comprises blending the mixed allergen product with one or morethan one probiotic selected from a group including, but not limited to,Lactobacillus species, Bifidobacterium species, Anacaerostipes species,and Bacillus species. For example, one or more probiotic may be selectedfrom a group including, but not limited to Lactobacillus rhamnosis,Anaerostipes caccae, Bifidobacterium longum, Bifidobacterium lactis,Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus brevis, Lactobacillus salivarius, Lactococcus lactis,Bacillus coagulans, Lactobacillus bulgaricus, Lactobacillus gasseri,Lactobacillus paracasei, Bacillus coagulans and Bifidobacterium bifidum.In a particular embodiment, a method of making a mixed allergen productfurther comprises blending the mixed allergen product with Lactobacillusrhamnosis and/or Bifidobacterium longum. As used herein, “probiotic”refers to a culture (pure or mixed), comprising microorganisms which,when ingested, beneficially affect the host.

In other embodiments, a method of making a mixed allergen productfurther comprises blending the mixed allergen product with one or morethan one prebiotic selected from a group including, but not limited to,fructooligosaccharide, inulin, isomalto-oligosaccharide, lactilol,lactosucrose, lactulose, pyrodextrin, soybean oligosaccharide,xylooligosaccharide, transgalactooligosaccharide, oligosaccharide,soluble corn fiber, beta-glucan, oligofructose, and bipolymer. In aparticular embodiment, a method of making a mixed allergen productfurther comprises blending the mixed allergen product withfructooligosaccharide. The term “prebiotic”, as used herein, refers to aspecific dietary food or nutrient that promotes the growth,proliferation, and/or activity of a specific, desirable, bacterialstrain.

A disclosed mixed allergen product may further include one or morevitamins, as desired. Vitamins that may be present include, for example,vitamin A (e.g., in an amount ranging from about 1 to about 35,000 IU),vitamin C (e.g., in an amount ranging from about 1 to about 1,000 mg),vitamin D (e.g., in an amount ranging from about 1 to about 4,000 IU,i.e. from about 0.025 to about 100 mcg), vitamin E (e.g., in an amountranging from about 1 to about 450 IU), vitamin K (e.g., in an amountranging from about 1 to about 250 μg), vitamin B-1 (thiamin; e.g., inamount ranging from about 1 to about 15 mg), vitamin B-2 (riboflavin;e.g., in an amount ranging from about 1 to about 17 mg), vitamin B-3(niacin; e.g., in an amount ranging from about 1 to about 200 mg),vitamin B-5 (pantothenic acid; e.g., in an amount ranging from about 1to about 100 mg), vitamin B-6 (pyridoxine; e.g., in an amount rangingfrom about 1 to about 30 mg), vitamin B-9 (folic acid; e.g., in anamount ranging from about 1 to about 4,000 μg), vitamin B-12 (cobalamin;e.g., in an amount ranging from about 1 to about 250 μg), vitamin H(biotin; e.g., in an amount ranging from about 1 to about 1,000 μg) andcombinations thereof. In certain embodiments, a mixed allergen productcomprises vitamin D. In certain embodiments, a mixed allergen productcomprises 400 IU, i.e. about 10 μg, of vitamin D.

Also contemplated is a method of making a mixed allergen product,wherein the mixed allergen product is further mixed with aphysiologically acceptable delivery vehicle to produce a physiologicallyacceptable composition. Mixed allergen products can be furtherincorporated into a variety of formulations for administration to asubject. More particularly, a mixed allergen product can be formulatedinto a physiological acceptable composition by combination withappropriate, physiologically acceptable carriers or diluents, forexample, a vegetable oil.

In certain embodiments, a disclosed mixed allergen product is designedfor oral immunotherapeutic treatment of food allergy in a child oradult, for example, as dispersible powders or granules, foods, tablets,troches, lozenges, gummies, emulsions, etc. Compositions intended fororal use may be prepared according to any convenient protocol for themanufacture of oral compositions and such compositions may contain oneor more agents selected from the group consisting of sweetening agents(e.g., glycerol, propylene glycol, sorbitol, or sucrose), flavoringagents, coloring agents and preserving agents in order to providepalatable preparations.

Also contemplated is a method of making a mixed allergen product,wherein the mixed allergen product is mixed or incorporated with food towhich a child or adult is not allergic. For example, foods may include,but are not limited to: baby or infant formula, baby food (e.g., pureedfood suitable for infant or toddler consumption), chips, cookies,breads, spreads, creams, yogurts, liquid drinks, chocolate containingproducts, candies, ice creams, cereals, coffees, pureed food products,etc.

Throughout the description, where apparatus, devices, and systems aredescribed as having, including, or comprising specific components, orwhere processes and methods are described as having, including, orcomprising specific steps, it is contemplated that, additionally, thereare apparatus, devices, and systems that consists essentially of, orconsist of, the recited components, and that there are processes andmethods that consist essentially of, or consist of, the recitedprocessing steps.

All examples presented herein are for illustrative purposes only, andshould not be construed as limiting in any way.

EXAMPLES Example 1

This example describes the selection of allergenic food ingredients forinclusion in an exemplary dry powder mixed allergen product containing16 individual complete food allergens (almond, cashew, hazelnut, peanut,pecan, pistachio, walnut, sesame, soy, egg, milk, oat, wheat, cod,salmon, and shrimp).

Ingredients were sourced with primary emphasis on domestic commercialavailability, with exceptions made for individual complete foodallergens that were only available internationally. Successfulcommercial sourcing of multiple options per each individual completefood allergen led to development of selection criteria in order tochoose the best commercial food allergen to be tested. Attributesscreened included: protein content; bulking material content;organoleptic attributes such as taste, presence of off-notes, andgrittiness; and solubility. Ingredients having considerably low proteincontent, or having a large proportion of bulking materials wereeliminated from contention. Individual complete food allergens weretasted dry to determine the presence of off-flavors and to assessgrittiness.

TABLE 1 shows a sample formulation for a mixed allergen product andrepresentative pre-weighed batch amounts of individual complete foodallergens provided for blending and sterilization.

TABLE 1 Individual Batch for Actual Complete Food FormulationFormulation Sterilization Batch Allergen (g) (%) Run (kg) (kg) AlmondPowder, 0.6895 6.620 33.10 33.10 50% Cashew Powder, 0.10720 10.294 51.4751.47 35% Cod Powder 0.02291 2.200 11.00 11.00 Whole Egg 0.06742 6.47332.37 32.37 Powder Hazelnut Powder 0.08824 8.472 42.36 42.36 MilkProtein 0.03534 3.393 16.96 6.35 Isolate Oat Protein 0.06266 6.017 30.0830.08 Peanut Powder 0.06122 5.879 29.39 29.39 Pecan Powder 0.09816 9.42547.13 47.13 Pistachio Powder 0.08867 8.514 42.57 42.57 Salmon Protein0.07560 7.259 36.30 36.30 Powder Sesame Seed 0.06100 5.857 29.28 29.28Powder Shrimp Powder 0.04261 4.092 20.46 20.46 Soy Protein 0.03736 3.58817.94 17.94 Isolate Walnut Powder 0.08316 7.985 39.93 39.93 Wheat Gluten0.04096 3.933 19.67 19.67 Total 1.04146 100.00 500.00 489.39

Example 2

This example describes an exemplary blending process for the preparationof a mixed allergen product comprising 16 individual complete foodallergens (almond, cashew, hazelnut, peanut, pecan, pistachio, walnut,sesame, soy, egg, milk, oat, wheat, cod, salmon, and shrimp).

A 1,000 kg ribbon blender (Prince Industries, India) was cleaned,sanitized, and tested for adenosine triphosphate (ATP) and allergens.Actual batch amounts (in kg) of 16 individual complete food allergens asindicated in Table 1 were loaded into the ribbon blender and blended at30 Hz for 15 minutes.

Bulk mixed allergen material was discharged from the ribbon blender intolined drums (40 to 50 kg per drum).

Example 3

This example describes an alternative blending and milling process forthe preparation of a mixed allergen product comprising 16 individualcomplete food allergens (almond, cashew, hazelnut, peanut, pecan,pistachio, walnut, sesame, soy, egg, milk, oat, wheat, cod, salmon, andshrimp).

All individual complete food allergens were weighed to make 8 kgbatches. Individual complete food allergens were transferred to a D500Hobart mixer equipped with a D-30 Bowl and safety cage. Individualcomplete food allergens were mixed with a paddle attachment “B” flatbeater on setting 1 for 2 minutes. Blended complete food allergens weretransferred to large, food-grade, plastic bags, sealed, and stored atroom temperature until milling. Blended complete food allergens wereslowly fed into a Quadro SLS-L1A FitzMill equipped with a #20 meshscreen and set to 9,000 rpm to achieve a target particle size of <500 μmin diameter. Milled complete food allergens were passed through a metaldetector and #35 mesh screen before packaging.

Example 4

A preliminary test for heat sensitivity was conducted to determine thesuitability of the bulk mixed allergen material for sterilizationtreatment.

Bulk mixed allergen material was placed in a pan and subjected todifferent temperatures in a laboratory oven for 5 minutes.

As shown in FIG. 1 , apart from a slight darkening in color withincreased temperature, bulk mixed allergen material did not exhibit anyother physical alterations with heat treatments of 95° C., 105° C., 110°C., or 120° C. for 5 minutes that would make the material unsuitable forsterilization treatment.

Example 5

This example describes an exemplary dry heat sterilization process thatcan be used for the preparation of a mixed allergen product comprising16 individual food allergens (almond, cashew, hazelnut, peanut, pecan,pistachio, walnut, sesame, soy, egg, milk, oat, wheat, cod, salmon, andshrimp). A dry heat sterilization method similar to that which iscontemplated in the present disclosure is described in US 2014/0023757,which is herein incorporated by reference.

Bulk mixed allergen material, blended as described in Examples 2 or 3,can be bagged in thermoresistant packaging. The bagged bulk mixedallergen material can then be placed in an insulated heat treatmentchamber, wherein heat is supplied to the treatment chamber by multiplenatural gas fired furnaces. Heat can be circulated through the insulatedtreatment chamber using multiple intake and exhaust ducts to facilitateuniform absorption of heat by the bagged bulk mixed allergen material.

For example, 6 bags of bulk mixed allergen material, each weighing about25 lbs./12 kg can be placed as a single layer on a pallet, and 14pallets can be loaded into a heating chamber having dimensions of 40feet long, 10 feet high, and 9 feet wide.

The heating chamber can then be sealed, and the chamber can be heated toreach the target temperature of 74° C. Warm-up time for the chamber toreach the target temperature may take up to about 24 hours.

The temperature of the heating chamber can be monitored using six probeswithin the heating chamber. For example, two probes may be placed in theback of the heating chamber, two probes may be place in the middle ofthe heating chamber, and two probes may be placed at the front of theheating chamber.

Once the target temperature is reached, the heating chamber can bemaintained for a period of 24 hours.

Temperatures of the insulated treatment chamber, multiple natural gasfired furnaces, and bagged bulk mixed allergen material can be monitoredto ensure that the bulk mixed allergen material is applied with dry heatsterilization at a temperature of about 65° C. to about 175° C. for aperiod of 24 hours.

Following dry heat treatment, the sterilized mixed allergen product canbe unloaded from the heating chamber. Water activity, moisture level,and total plate counts can be measured for samples of the sterilizedmixed allergen product and compared to samples of the bulk mixedallergen material prior to dry heat sterilization.

For example, samples can be diluted and spread onto petri dishes ofgeneral recovery media to measure colony-forming units per gram ofproduct or material (CFU/g) for total aerobic microorganism andEnterobacteriaceae counts. E. coli/coliform, and S. aureus counts can bedetermined by diluting samples and spreading onto 3M Petrifilm™ E.coli/coliform and 3M Petrifilm™ Staph Express count plates,respectively, in accordance with the manufacturer's instructions (3M,St. Paul, Minnesota).

Samples can be additionally tested for the presence of Listeria andSalmonella using the BAX® PCR detection system (Hygiena, Camarillo,California) in accordance with the manufacturer's instructions.

Total mold and yeast counts can also be determined by diluting samplesand spreading onto 3M Petrifilm™ mold and yeast count plates inaccordance with the manufacturer's instructions.

Example 6

This example describes the validation of a dry heat process forsterilization of bulk mixed allergen material.

In this dry heat process, SurroNov® 1830, a strain derived fromEnterococcus Faecium ATCC®8459, was used as a surrogate microorganism toevaluate the lethality of the dry heat sterilization on Salmonella andCronobacter pathogens. SurroNov® 1830 has a thermal resistance similarto, or greater than, the thermal resistance of Salmonella andCronobacter. Validation was aimed at achieving at least a 5-logreduction in the number of viable SurroNov® 1830 organisms. Twovalidation trials were performed to evaluate variability.

Before dry heat sterilization, 70 thermoresistant bags, each containing25 g of bulk mixed allergen material blended as described in Examples 2or 3, were inoculated with approximately 1.4×10⁶ SurroNov®1830organisms. Each inoculated thermoresistant bag was placed into a box.

Six inoculated bags of bulk mixed allergen material were not introducedinto the dry heat sterilization process and served as non-treatedcontrol samples.

14 pallets, each containing four boxes of inoculated thermoresistantbags of bulk mixed allergen material, were loaded into the heatingchamber as shown in FIGS. 2A and 2B for validation trials 1 and 2,respectively. On pallets 1, 6, 9, and 14, second inoculatedthermoresistant bags of bulk mixed allergen material were placed intoboxes 3 and/or 4.

Temperature of the heating chamber was monitored throughout thevalidation run using heat chamber probes: two positioned at the front ofthe heating chamber, two positioned in the middle of the heatingchamber, and two positioned at the back of the heating chamber.Additionally, MadgeTech probes were included to monitor temperature ofthe heating chamber during the dry heat sterilization process. MadgeTechprobes were either placed inside select inoculated thermoresistant bagsof bulk mixed allergen material or outside the boxes.

As shown in FIGS. 3A and 3B for validation trials 1 and 2, respectively,the temperature recorded by the MadgeTech probes are relativelyhomogeneous at different positions inside the chamber. Temperaturesrecorded by the heat chamber probes differ depending on the positioninside the chamber (i.e. back, middle, or front), but temperaturesbetween two probes in the same position are relatively similar duringthe dry heat sterilization treatment.

For validation trial 1 (FIG. 3A), the dry heat treatment was initiallyset to 175° F./80° C. It took 18 hours for all probes to reach thetarget temperature. Once the target temperature was attained, thetemperature was decreased to 165° F./74° F. and maintained at thistemperature for an additional 24 hours.

For validation trial 2 (FIG. 3B), the dry heat treatment was set to 165°F./74° C. It took 17 hours for all probes to reach the targettemperature. Once the target temperature was attained, the dry heatsterilization temperature was maintained at 165° F./74° C. for anadditional 24 hours.

As shown in TABLE 2, water activity and moisture content of sterilizedmixed allergen product after validation trial 1 dry heat sterilizationis lower than the water activity and moisture content of bulk mixedallergen material before dry heat treatment. Total plate counts onselective media were similar for bulk mixed allergen material before dryheat sterilization and mixed allergen product after dry heatsterilization.

TABLE 2 Water Activity Moisture Log Total Plate (a_(w)) Content (%)Count Bulk Mixed Allergen 0.286 ± 0.002 1.63 ± 0.08 % 3.0 ± 0.3 logcfu/g Material (before dry heat sterilization) Mixed Allergen 0.235 ±0.029 1.26 ± 0.21 % 2.8 ± 0.7 log cfu/g Product (after dry heatsterilization)

As shown in TABLE 3, bulk mixed allergen material samples that were nottreated with dry heat sterilization had a mean SurroNov® 1830 count of8.1±0.1 log cfu/g.

TABLE 3 Control Sample CFU/g Log Mean 1 1.4 × 10⁸ 8.1 8.1 ± 0.1 2 1.5 ×10⁸ 8.2 3 1.5 × 10⁸ 8.2 4 1.2 × 10⁸ 8.1 5 1.3 × 10⁸ 8.1 6 1.5 × 10⁸ 8.2

As shown in TABLE 4, greater than 5 log reductions in SurroNov® 1830CFU/g was achieved in all samples following dry heat sterilization invalidation trial 1, as compared to inoculated bulk mixed allergenmaterial samples that were not dry heat treated.

TABLE 4 ID CFU/g Log Log Reduction PS7 <10 <1 >7.1 PS8 <10 <1 >7.1 PS9<10 <1 >7.1 PS10 <10 <1 >7.1 PS11 <10 <1 >7.1 PS12 <10 <1 >7.1 PS13 <10<1 >7.1 PS14 <10 <1 >7.1 PS15 <10 <1 >7.1 PS16 <10 <1 >7.1 PS17 <10<1 >7.1 PS18 <10 <1 >7.1 PS19 <10 <1 >7.1 PS20 <10 <1 >7.1 PS21 <10<1 >7.1 PS22 <10 <1 >7.1 PS23 <10 <1 >7.1 PS24 <10 <1 >7.1 PS25 <10<1 >7.1 PS26 <10 <1 >7.1 PS27 <10 <1 >7.1 PS28 <10 <1 >7.1 PS29 <10<1 >7.1 PS30 <10 <1 >7.1 PS31 <10 <1 >7.1 PS32 <10 <1 >7.1 PS33 <10<1 >7.1 PS34 <10 <1 >7.1 PS35 <10 <1 >7.1 PS36 <10 <1 >7.1 PS37 <10<1 >7.1 PS38 <10 <1 >7.1 PS39 <10 <1 >7.1 PS40 <10 <1 >7.1 PS41 <10<1 >7.1 PS42 <10 <1 >7.1 PS43 <10 <1 >7.1 PS44 <10 <1 >7.1 PS45 <10<1 >7.1 PS46 <10 <1 >7.1 PS47 <10 <1 >7.1 PS48 <10 <1 >7.1 PS49 410 2.65.5 PS50 20 1.3 6.8 PS51 <10 <1 >7.1 PS52 <10 <1 >7.1 PS53 <10 <1 >7.1PS54 <10 <1 >7.1 PS55 <10 <1 >7.1 PS56 10 1 7.1 PS57 <10 <1 >7.1 PS58<10 <1 >7.1 PS59 <10 <1 >7.1 PS60 <10 <1 >7.1 PS61 <10 <1 >7.1 PS62 <10<1 >7.1 PS63 <10 <1 >7.1 PS64 <10 <1 >7.1 PS65 <10 <1 >7.1 PS66 <10<1 >7.1 PS67 <10 <1 >7.1 PS68 <10 <1 >7.1 PS69 <10 <1 >7.1 PS70 <10 <1>7.1

As shown in TABLE 5, water activity of sterilized mixed allergen productafter validation trial 2 dry heat sterilization is similar to the wateractivity of bulk mixed allergen material before dry heat treatment.Moisture content and total plate counts were lower following dry heatsterilization treatment in validation trial 2, as compared to bulk mixedallergen material that was not dry heat sterilized.

TABLE 5 Water Moisture Log Total Plate Activity (aw) Content (%) CountBulk Mixed Allergen 0.285 ± 0.005 1.95 ± 0.10 % 3.0 ± 0.3 log cfu/gMaterial (before dry heat sterilization) Mixed Allergen 0.301 ± 0.0541.22 ± 0.12 % 2.5 ± 0.1 log cfu/g Product (after dry heat sterilization)

As shown in TABLE 6, bulk mixed allergen material samples that were nottreated with dry heat sterilization had a mean SurroNov® 1830 count of8.1±0.1 log cfu/g.

TABLE 6 Control Sample CFU/g Log Mean 1 1.5 × 10⁸ 8.2 8.1 ± 0.1 2 1.3 ×10⁸ 8.1 3 1.1 × 10⁸ 8.0 4 1.1 × 10⁸ 8.0 5 6.9 × 10⁷ 7.8 6 1.3 × 10⁸ 8.17 1.1 × 10⁸ 8.0 8 1.4 × 10⁸ 8.1

Similarly to dry heat sterilization in validation trial 1, as shown inTABLE 7, dry heat sterilization in validation trial 2 resulted ingreater than 5 log reductions in SurroNov® 1830 CFU/g in all samples, ascompared to inoculated bulk mixed allergen material samples that werenot dry heat treated.

TABLE 7 ID CFU/g Log Log Reduction PS7 <10 <1 >7.1 PS8 <10 <1 >7.1 PS9<10 <1 >7.1 PS10 <10 <1 >7.1 PS11 <10 <1 >7.1 PS12 <10 <1 >7.1 PS13 <10<1 >7.1 PS14 <10 <1 >7.1 PS15 <10 <1 >7.1 PS16 <10 <1 >7.1 PS17 <10<1 >7.1 PS18 <10 <1 >7.1 PS19 <10 <1 >7.1 PS20 <10 <1 >7.1 PS21 <10<1 >7.1 PS22 <10 <1 >7.1 PS23 <10 <1 >7.1 PS24 <10 <1 >7.1 PS25 <10<1 >7.1 PS26 20 1.3 6.8 PS27 <10 <1 >7.1 PS28 <10 <1 >7.1 PS29 <10<1 >7.1 PS30 <10 <1 >7.1 PS31 <10 <1 >7.1 PS32 <10 <1 >7.1 PS33 <10<1 >7.1 PS34 <10 <1 >7.1 PS35 <10 <1 >7.1 PS36 <10 <1 >7.1 PS37 20 1.36.8 PS38 <10 <1 >7.1 PS39 <10 <1 >7.1 PS40 <10 <1 >7.1 PS41 <10 <1 >7.1PS42 <10 <1 >7.1 PS43 <10 <1 >7.1 PS44 <10 <1 >7.1 PS45 <10 <1 >7.1 PS46<10 <1 >7.1 PS47 <10 <1 >7.1 PS48 <10 <1 >7.1 PS49 410 2.6 5.5 PS50 201.3 6.8 PS51 <10 <1 >7.1 PS52 <10 <1 >7.1 PS53 <10 <1 >7.1 PS54 <10<1 >7.1 PS55 <10 <1 >7.1 PS56 10 1 7.1 PS57 <10 <1 >7.1 PS58 <10 <1 >7.1PS59 <10 <1 >7.1 PS60 <10 <1 >7.1 PS61 <10 <1 >7.1 PS62 <10 <1 >7.1 PS63<10 <1 >7.1 PS64 <10 <1 >7.1 PS65 <10 <1 >7.1 PS66 <10 <1 >7.1 PS67 <10<1 >7.1 PS68 <10 <1 >7.1 PS69 <10 <1 >7.1 PS70 <10 <1 >7.1

Example 7

Particle size distribution can be measured for mixed allergen productsamples treated with dry heat sterilization and compared to theparticles of corresponding bulk mixed allergen material that did notreceive application of dry heat sterilization. Samples can be filteredfor 3 minutes using a Ro-Tap® sieve shaker and a series of mesh sizesconsisting of #10, #12, #14, #16, #18, #20, #25, #30, #35, #40, and #50meshes. Filtered particles and particles retained by each mesh can beindividually removed, weighed, and distributions plotted as % weightversus particle size (in m).

Example 8

Protein integrity of mixed allergen product samples treated with dryheat sterilization can be compared to bulk mixed allergen material thatdid not receive application of dry heat sterilization. Protein integritycan be assessed by resolving all proteins present in non-sterilized anddry heat-sterilized samples by SDS-PAGE.

1 mg of bulk mixed allergen material or dry heat sterilized mixedallergen product can be weighed into 2 mL microtubes. Based on theirassessed protein concentrations, samples can be diluted with Laemmlibuffer containing 5% 2-mercaptoethanol (BioRad, Hercules, California) toa protein concentration of 1 mg/ml. Protein suspensions can be mixed bygentle inversion or intermittent vortexing for 5 minutes then heated to95° C. to 100° C. for 5 minutes. Samples can be centrifuged for 1 minuteat 1,000 RPM, then 10 μl of each sample can be loaded per well, intriplicate, on a 4-20% polyacrylamide gradient gel (BioRad, Hercules,California) immersed in 1×SDS-PAGE running buffer (BioRad, Hercules,California). Gels can be run at 100 V for 80-90 minutes (or 200V for30-40 minutes). Gels can then be washed 3 times with distilled water, 5minutes per wash, on an orbital shaker at room temperature. Gels can befixed in fixing solution (Sigma-Aldrich, St. Louis, Missouri) or 50%methanol, 10% acetic acid, for 15 minutes. Gels can be stained with EXBlue gel staining reagent (Sigma-Aldrich, St. Louis, Missouri) for 2hours or overnight, then de-stained by washing in distilled water for1-2 hours or overnight. Upon completion of washing, gels can be coveredwith clear plastic and scanned using a gel reader. Protein banddensitometry can be analyzed using ImageJ public domain software (NIH,Bethesda, Maryland).

Example 9

The visual and organoleptic characteristics of mixed allergen productsamples treated with dry heat sterilization can be compared to bulkmixed allergen material that did not receive application of dry heatsterilization. A comparison of taste profiles of dry heat sterilizedmixed allergen product and non-sterilized bulk mixed allergen can alsobe performed.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety for all purposes. In case of conflict, thepresent application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

1. A method of making a mixed allergen product substantially free ofreplication viable organisms, comprising: providing bulk mixed allergenmaterial comprising 2 to 20 individual complete food allergens; andsterilizing the bulk mixed allergen material by dry heat sterilization,thereby obtaining the mixed allergen product.
 2. The method of claim 1,further comprising providing 2 to 20 individual complete food allergens,and blending the 2 to 20 individual complete food allergens together toobtain the bulk mixed allergen material, wherein: the individualcomplete food allergens are selected from the group consisting ofalmond, cashew, cod, egg, hazelnut, milk, oat, peanut, pecan, pistachio,salmon, sesame, shrimp, soy, walnut, and wheat; and/or blending furthercomprises blending the 2 to 20 individual complete food allergens withone or more than one bulking agent; and/or after blending, the bulkmixed allergen material is discharged into lined drums. 3-6. (canceled)7. The method of claim 1, wherein the dry heat sterilization is at atemperature of about 65° C. to about 175° C. for about 18 hours to about72 hours, wherein: the dry heat sterilization comprises using asterilization system having a furnace and a heating chamber, wherein thefurnace provides about 250,000 BTUs to about 750,000 BTUs of heat to theheating chamber; and/or the bulk mixed allergen material is firstpackaged into thermoresistant packaging before sterilizing; and/ortemperature probes are placed with the bulk mixed allergen materialduring sterilizing to monitor the temperature of the dry heatsterilization. 8-10. (canceled)
 11. The method of claim 1, wherein themixed allergen product has at least about a 1 log reduction to at leastabout a 4 log reduction in aerobic bacterial organism plate counts ascompared to corresponding bulk mixed allergen material that has not beensterilized.
 12. The method of claim 1, wherein the mixed allergenproduct has: less than about 100,000 CFU/g of aerobic bacterialorganisms, and/or less than about 10 CFU/g of coliforms, and/or lessthan about 10 CFU/g of Escherichia coli, and/or less than about 10 CFU/gof Enterobacteriaceae, and/or less than about 10 CFU/g of Salmonella,and/or less than about 10 CFU/g of Cronobacter, and/or less than about10 CFU/g of Staphylococcus aureus, and/or less than about 10 CFU/g ofyeast, and/or less than about 10 CFU/g of mold. 13-17. (canceled) 18.The method of claim 1, wherein the mixed allergen product is negative ina PCR-based assay for Listeria species or Cronobacter species per 25grams of the mixed allergen product.
 19. (canceled)
 20. The method ofclaim 1, wherein the mixed allergen product is negative in a PCR-basedassay for Salmonella species per 375 g of the mixed allergen product.21. (canceled)
 22. (canceled)
 23. The method of claim 1, wherein themixed allergen product has a fat content of about 10% to about 20% byweight.
 24. The method of claim 1, wherein protein integrity of themixed allergen product is substantially similar to protein integrity ofcorresponding bulk mixed allergen material that has not been sterilized.25. The method of claim 24, wherein the protein integrity of the mixedallergen product is determined by SDS-PAGE.
 26. The method of claim 1,wherein the mixed allergen product has a protein content of about 40% toabout 50% by weight.
 27. The method of claim 1, wherein the mixedallergen product has about 1% to about 10% moisture by weight.
 28. Themethod of claim 1, wherein the mixed allergen product has a wateractivity of about 0.1 to about 0.6.
 29. The method of claim 1, whereinless than about 10% by weight of the mixed allergen product areparticles having a diameter greater than 500 μm.
 30. The method of claim1, wherein the mixed allergen product is darker in color as compared tocorresponding bulk mixed allergen material that has not been sterilized.31. The method of claim 1, wherein the mixed allergen product has asubstantially similar taste profile as compared to corresponding bulkmixed allergen material that has not been sterilized.
 32. The method ofclaim 1, wherein sterilizing further comprises one or more than onefurther application of dry heat sterilization.
 33. A method of making amixed allergen product substantially free of replication viableorganisms, comprising: providing 2 to 20 individual complete foodallergens; sterilizing each of the 2 to 20 individual complete foodallergens by dry heat sterilization, thereby obtaining 2 to 20sterilized individual complete food allergens; and blending the 2 to 20sterilized individual complete food allergens together, therebyobtaining the mixed allergen product. 34-40. (canceled)
 41. A mixedallergen product substantially free of replication viable organismsprepared by the method of claim 1.